Proteins are dynamic molecular machines, undergoing motions on a wide range of time scales. Although there is considerable evidence both from theory and experiment that many enzymes are inherently flexible, the fundamental question of how, or even if, protein fluctuations couple to catalytic function remains unanswered. Are protein motions coupled to the chemical transformation, or are they involved primarily in controlling the flux of substrate, products, or cofactors? What is the time scale of active site conformational changes required for catalysis? How is the energy landscape of the enzyme modulated during the catalytic cycle? These and other issues will be addressed using state-of-the-art NMR methods to elucidate the dynamic properties of an exceptionally well- characterized enzyme, dihydrofolate reductase from E. coli, in all of the intermediate states formed along its reaction pathway. DHFR is the target for anti-folate drugs such as the anticancer agent methotrexate and the antibacterial trimethoprim and is of major biomedical significance. The proposed research will focus on characterization of microsecond-millisecond time scale fluctuations in the active site, on the same time scale as key events in DHFR catalysis. The NMR experiments will provide a detailed dynamic and thermodynamic description of slow motions of the active site loops in all intermediate states involved in the catalytic cycle and in carefully selected mutants that impair the catalytic process. These experiments build upon earlier research from this laboratory that mapped the active site conformational states of DHFR and its fast (ps-ns) time scale dynamics. The proposed research will provide novel insights into the coupling between protein motions and catalytic function in DHFR, as well as an understanding of how these motions are modulated by interaction with substrate, cofactor, and products at various stages in the catalytic cycle.